Engineering improved specificity and activity into oral bacterial sialidases for glycan biotechnology applications

研究设计和方法学1-PG 0352和NanH的结构理解（Yr 1-2）鉴于PG 0352和NanH唾液酸酶的新活性和CBM结构域，需要它们的结构信息来理解作用模式和指导工程。- 使用GS和JR建立的方案，我们将纯化和结晶完整的NanH和PG 0352及其分离的CBM结构域（其中唾液缀合物将不被切割），其为载脂蛋白和配体结合形式（例如3/6-S-乳糖、SLeA/X、S-Tn、DANA）。我们有条件结晶的NanH CBM（GS，JR），这表明其他构建体的良好潜力。为了增加新配体结合信息的机会，我们将产生无活性版本（FRIP突变）以促进配体结合。同时，我们将基于pdb-hit信息和在此获得的信息对NanH和PG 0352进行深入的生物信息学和建模研究，以预测可能提高特异性的残基。2-挖掘口腔微生物组中的新型唾液酸酶（Yr 1-2）人类口腔微生物组数据库资源的初步筛选揭示了大量具有新潜力的唾液酸酶。- 我们将挖掘这一资源和一系列口服宏基因组数据集，以获得新型唾液酸酶，我们将生产作为密码子优化（新型IP）克隆的唾液酸酶，我们将使用我们建立的糖缀合物（GS）和糖蛋白（DS）去唾液酸化测定法（使用标准生物化学方法（硫代巴比妥酸测定法（GS））或衍生的HPLC或MS方法（DS-在Ludger进行）纯化和评估活性。这些也将进入结晶试验，以增加成功解决结构问题的机会。3-工程化唾液酸酶以提高特异性和活性（Yr 2-3）-基于建模、结构信息（1）和催化性精氨酸三联体和特征性FRIP结构域和Asp-box的保守性，我们将靶向活性位点周围的残基（例如T. cruzi酶）和CBM接触配体的配体结合残基。我们将能够实现这一点的可能性增加了我们的新方法，检查CBM结构域的隔离，也在使用的失活突变体，应该仍然结合配体。

To understand and develop orally derived sialidases for the glycobiology industry.Study design and methodology1-Structural understanding of PG0352 and NanH (Yr 1-2)Structural information on PG0352 and NanH sialidases is needed given their novel activity and CBM domains to understand mode of action and direct engineering. -Using protocols established by GS and JR we will purify and crystallise full NanH and PG0352 and their isolated CBM domains (where sialloconjugates will not be cleaved) in apo- and ligand-bound forms (e.g. 3/6- S-lactose, SLeA/X, S-Tn, DANA). We have conditions for crystallization of the NanH CBM (GS, JR) suggesting good potential for other constructs. To increase chances of novel ligand bound information we will produce inactive versions (FRIP mutations) to promote ligand binding.-In parallel we will undertake in-depth bioinformatic and modeling studies of NanH and PG0352 based on pdb-hit information and that gained here to predict residues that might improve specificity.2-Mining of the oral microbiome for novel sialidases (Yr 1-2)Preliminary screening of the Human Oral Microbiome Database resource reveals a plethora of sialidases with novel potential. - We will mine this resource and a range of oral metagenome datasets for novel sialidases that we will produce as codon optimized (novel IP) clones that we will purify and assess for activity using our established glyconjugate (GS) and glycoprotein (DS) desialylation assays using standard biochemical methods (Thiobarbiturate assay (GS)) or derivatised HPLC or MS based methods (DS- to be performed at Ludger). These will also enter crystallisation trials to increase the opportunities for successful structure solution.3- Engineering sialidases for specificity and activity improvements (Yr 2-3)-Based on modeling, structural information (1) and on the conservation of the catalytic Arginine triad and characteristic FRIP domain and Asp-boxes we will target residues surrounding the active site (e.g. Trp212 and Tyr119 of the T. cruzi enzyme) and ligand-binding residues of the CBM contacting ligands. The likelihood that we will be able to achieve this is increased by our novel approach examining the CBM domains in isolation and also in the use of inactive mutants that should still bind ligands.